The present invention generally relates to somatotropins. More particularly, the present invention relates to non-denaturing methods for separating biologically active somatotropin protein fractions from somatotropin samples in a manner suitable to provide potency assays.
The production of bovine somatotropin (bST) and other somatotropins in large scale has recently been fostered by recombinant DNA technology. For example, recombinant microorganisms such as recombinant Escherichia coli produce insoluble granules of bST in their cytoplasm. These granules, known as refractile bodies or inclusion bodies, contain aggregated denatured somatotropin. The refractile bodies are recovered and usually treated with a denaturant such as guanadine hydrochloride, sodium dodecyl sulfate or urea. The denaturant unfolds and solubilizes the improperly folded bST molecules. Afterward, the bST molecules are renatured to form the properly folded, biologically active bST monomeric protein.
Due to inefficiencies of these denaturing and renaturing steps, however, some aggregates of improperly folded, biologically inactive somatotropin aggregates are also formed. Thus, the bST or other similar somatotropin bulk material obtained contains both the biologically active monomer and the biologically inactive aggregates. It is therefore important to establish a method for determining the potency of bST and other somatotropins in quality control.
The potency of bST and other somatotropins has previously been estimated by a rat weight gain method. Essentially, the weight gain of rats to which somatotropin samples are administered is monitored, and from this data a value representing the potency of the somatotropin is obtained. However, this assay cannot be employed to accurately quantify the somatotropins in routine analysis. Somatotropin potency has also been determined by radio receptor assay (RRA). However, RRA is time consuming. Also, RRA is inaccurate, and thus several tests are usually performed and the results averaged to provide a potency value.
Reversed-phase high performance liquid chromatography (RPHPLC) has been employed to determine proteins. However, most RPHPLC methods which have been used are not appropriate candidates for measuring the potency of bST because they have employed acidic mediums and organic solvents in the mobile phase, which denature bST.
Size exclusion high performance liquid chromatography ("size exclusion HPLC") using a mobile phase containing sodium dodecyl sulfate or guanadine hydrochloride has previously been employed to determine bST. However, such methods are not bio-potency assays because their mobile phases are denaturing to bST.
What is therefore needed are expedient, precise and accurate non-denaturing assays for determining the bio-potency of somatotropins. The present invention addresses these needs.